Colorectal cancer (CRC) is the third leading cause of cancer deaths in the US. Most deaths are the result of metastatic disease for which treatment options are limited. Thus, there is an urgent need for a better understanding of the biological processes driving metastasis and for new therapies. To this end, we, and our collaborators have identified KCNQ1, a member of the Kv7 potassium channel subfamily, as a novel CRC tumor suppressor gene in mice and humans with potential clinical usefulness. In the ApcMin background, Kcnq1-deficient mice show an increased incidence of adenomas that in some cases progress to adenocarcinomas. More importantly, in human CRC, elevated expression of KCNQ1 protein is associated with improved survival in patients undergoing resection for Stage IV colorectal cancer liver metastases with a highly significant increase in median overall survival of 23 months. We have very recently confirmed this finding in earlier stage CRC, where maintenance of KCNQ1 expression was associated with a significant increase in disease-free and overall survival in stage II & stage III CRC. Accordingly, our goal long-term goal is to determine if KCNQ1 can be used clinically as a biomarker or therapeutic target. In this proposal we will investigate the cellular mechanisms that underlie the tumor suppressor phenotype of KCNQ1 in CRC by employing intestinal organoid cultures as CRC disease surrogates. CRC development & progression likely arises from dysregulation of the stem cell compartment in the base of the intestinal crypt. The intestinal crypt can be modeled in vitro by 3D organoid culture as intestinal organoids recapitulate normal crypt morphology and physiological function, and thus, can serve as valuable tissue surrogates. In particular, alterations to the stem cell compartment are reflected in changes in organoid survival, growth and morphology. In our preliminary work we observed that Kcnq1 deficiency in mice resulted in a three-fold increase in intestinal organoid outgrowth, along with a significant delay in organoid differentiation, thus, potentially, directly linking Kcnq1 to the intestinal epithelial stem cell compartment. Therefore, based on these observations, the central hypothesis of this proposal is that KCNQ1 protects against CRC progression by regulating the intestinal stem cell compartment, a process that can be efficiently modeled using organoid cultures. In Specific Aim 1 we will use organoid culture to investigate the role of Kcnq1 in regulation of the stem cell compartment, tumor progression and metastasis in mouse models. In Specific Aim 2 we will examine the role of KCNQ1 in late stage human CRC by directly examining organoid cultures derived from human colorectal cancer liver metastases and by examining their metastatic potential when engrafted into immunodeficient mice. If KCNQ1 is found to regulate the stem cell compartment, this result will substantiate a clinically significant role for KCNQ1 in human colorectal cancer and form the basis for further studies to determine the molecular pathways linking KCNQ1 activity to stem cell capacity and CRC processes.